Biochimica et Biophysica Acta 1826 (2012) 443–457

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbacan

Review

Ascorbic acid: Chemistry, biology and the treatment of cancer☆

Juan Du a, Joseph J. Cullen a, b, c, d, Garry R. Buettner a, c,⁎
a
Department of Radiation Oncology, University of Iowa College of Medicine, Iowa City, IA, USA
b
Department of Surgery, University of Iowa College of Medicine, Iowa City, IA, USA
c
Holden Comprehensive Cancer Center, USA
d
Veterans Affairs Medical Center, Iowa City, IA, USA

a r t i c l e i n f o a b s t r a c t

Article history: Since the discovery of vitamin C, the number of its known biological functions is continually expanding. Both
Received 1 March 2012 the names ascorbic acid and vitamin C reflect its antiscorbutic properties due to its role in the synthesis of
Received in revised form 11 June 2012 collagen in connective tissues. Ascorbate acts as an electron-donor keeping iron in the ferrous state thereby
Accepted 13 June 2012
maintaining the full activity of collagen hydroxylases; parallel reactions with a variety of dioxygenases affect
Available online 20 June 2012
the expression of a wide array of genes, for example via the HIF system, as well as via the epigenetic land-
Keywords:
scape of cells and tissues. In fact, all known physiological and biochemical functions of ascorbate are due to
Ascorbate its action as an electron donor. The ability to donate one or two electrons makes AscH − an excellent reducing
Oxidative stress agent and antioxidant. Ascorbate readily undergoes pH-dependent autoxidation producing hydrogen perox-
Hydrogen peroxide ide (H2O2). In the presence of catalytic metals this oxidation is accelerated. In this review, we show that the
Cancer chemical and biochemical nature of ascorbate contribute to its antioxidant as well as its prooxidant proper-
Pharmacology ties. Recent pharmacokinetic data indicate that intravenous (i.v.) administration of ascorbate bypasses the
Nutrition tight control of the gut producing highly elevated plasma levels; ascorbate at very high levels can act as
prodrug to deliver a significant flux of H2O2 to tumors. This new knowledge has rekindled interest and
spurred new research into the clinical potential of pharmacological ascorbate. Knowledge and understanding
of the mechanisms of action of pharmacological ascorbate bring a rationale to its use to treat disease especial-
ly the use of i.v. delivery of pharmacological ascorbate as an adjuvant in the treatment of cancer.
Published by Elsevier B.V.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
2. Chemistry and biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
3. Biosynthesis and uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
4. Biological functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
4.1. Ascorbate as enzyme cofactors of hydroxylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
4.2. Ascorbate as a cofactor in the HIF system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
4.3. Ascorbate as a cofactor in histone demethylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
4.4. Ascorbate and cancer prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
5. Recycling of ascorbate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
5.1. Reduction of ascorbate free radical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
5.2. Reduction of dehydroascorbate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
6. Ascorbate as an antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
7. Pro-oxidant effects of ascorbate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448

Abbreviations: Asc, a general abbreviation to include all forms, i.e. AscH2 + AscH− + Asc2 − + DHA; AscH2, ascorbic acid, vitamin C; AscH−, ascorbate monoanion; Asc2 −, ascor-
bate dianion; Asc•−, semidehydroascorbate, i.e. ascorbate radical; DHA, dehydroascorbic acid; HIF, hypoxia inducible factor; PDI, protein disulfide isomerase; SVCT,
sodium-dependent vitamin C transporters
☆ Supported by NIH grants CA137230 and 1R01GM073929, the Medical Research Service, Department of Veterans Affairs, and the Susan L. Bader Foundation of Hope.
⁎ Corresponding author at: Department of Radiation Oncology, Free Radical and Radiation Biology, Med Labs B180, University of Iowa College of Medicine, Iowa City, IA 52242,
USA. Tel.: +1 319 335 8015 (office), +1 319 335 8019 (secretary); fax: +1 319 335 8039.
E-mail address: garry-buettner@uiowa.edu (G.R. Buettner).

0304-419X/$ – see front matter. Published by Elsevier B.V.

doi:10.1016/j.bbcan.2012.06.003
444 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457

8. Ascorbate and cancer treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449

8.1. The role of hydrogen peroxide generation and removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
8.2. Ascorbate-induced cytotoxicity in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
8.3. High dose ascorbate in animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
8.4. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
8.5. Ascorbate and conventional cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
9. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
10. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

1. Introduction Ascorbate oxidizes readily. The rate of oxidation is dependent on pH

In the 80 years since the discovery of vitamin C (ascorbic acid, AscH2;
ascorbate, AscH−) [1,2], the number of its known biological functions is
continually expanding. Because of the ease of oxidation of ascorbate,
gaining the first understanding of its role as the antiscorbutic vitamin
was a major challenge. As a water-soluble reducing agent and donor an-
tioxidant, AscH− can undergo two consecutive, one-electron oxidations
resulting in the formation of ascorbate radical (Asc•−) and dehy-
droascorbic acid (DHA) (Fig. 1). Ascorbate can be regenerated from ascor-
bate radical and DHA, either enzymatically or non-enzymatically.
Ascorbate readily undergoes pH-dependent autoxidation producing hy-
drogen peroxide [3]. In the presence of catalytic metals this oxidation is
accelerated [4]. Ascorbate can also have pro-oxidant effects. In fact the
combination of iron and ascorbate has long been used as an oxidizing sys-
tem; the combination of these two reagents is referred to as the
Udenfriend system and is used for the hydroxylation of alkanes, aro-
matics, and other oxidations [5,6]. In addition, ascorbate serves as a reduc-
ing cofactor for many enzymes, for example hydroxylases that belong to
the Fe2+–2-oxoglutarate-dependent families of dioxygenases.
The uptake of ascorbate from the intestinal tract is very tightly
controlled [7]. However, recent pharmacokinetic data indicate that
intravenous administration of ascorbate can bypass this tight control
resulting in highly elevated plasma levels [8]. Because ascorbate read-
ily oxidizes to produce H2O2, pharmacological ascorbate has been
proposed as a prodrug for the delivery of H2O2 to tumors [9–12].
This new knowledge brings insights into the controversial role of
ascorbate in the treatment of cancer. In this review we present the
fundamental chemistry and biochemistry of ascorbate that may
have a role in the mechanisms of high dose, i.v. ascorbate in the treat-
ment of cancer.
2. Chemistry and biochemistry
Ascorbic acid (AscH2, vitamin C) is a water-soluble ketolactone
with two ionizable hydroxyl groups. It has two pKa's, pK1 is 4.2 and
pK2 is 11.6; thus, the ascorbate monoanion, AscH −, is the dominant
form at physiological pH (Fig. 1). Ascorbate is an excellent reducing
agent and readily undergoes two consecutive, one-electron oxida-
tions to form ascorbate radical (Asc •−) and dehydroascorbic acid
(DHA). The ascorbate radical is relatively unreactive due to resonance
stabilization of the unpaired electron; it readily dismutes to ascorbate
and DHA (kobs = 2 × 10 5 M −1 s −1, pH 7.0) [4].
•− þ −
2Asc þ H ↔AscH þ DHA:
These properties make ascorbate an effective donor antioxidant
[13].
Pure ascorbic acid is a white crystalline powder, extremely soluble
in water making a colorless solution. Sodium ascorbate often contains
significant quantities of oxidation products departing a yellow color.
At pH values between 6 and 7.8, the purity and concentration of an
ascorbate solution can be easily determined by its absorbance,
ε265 = 14,500 M −1 s −1 [14].
and is accelerated by catalytic metals [14]. In the absence of catalytic
metals, the spontaneous oxidation of ascorbate is quite slow at pH 7.0
[15]. This autoxidation, i.e. oxidation in the absence of catalytic metals,
occurs via the ascorbate dianion, Asc 2− [16]. At pH 7.0 the dominant
species for vitamin C is AscH− (99.9%) with low concentrations of
AscH2 (0.1%) and Asc 2− (0.005%). The amount of Asc 2− will increase
by a factor of ten with a one unit increase in pH; this will increase
the rate of autoxidation by a factor of 10. In an air-saturated phosphate
buffer (pH 7.0), it has been estimated that the observed pseudo-
first-order rate constant for the autoxidation of ascorbate is on the
order of 10−7–10−6 s−1 at pH 7.0 [15], consistent with a rate constant
of ≈300 M−1 s −1 for the true autoxidation of Asc2−, Reaction 2 [17].
k ¼ 300 M−1 s−1 ð2Þ
2− •− •−
Asc þ O2 →Asc þ O2 :
Thus, true autoxidation is indeed very slow. In most laboratory set-
tings, oxidation of ascorbate is catalyzed by adventitious catalytic metals
in the buffers as well as metals that enter the buffer for laboratory equip-
ment [15,18]. In near-neutral buffers with contaminating metals, the ox-
idation and subsequent loss of ascorbate can be very rapid.
3. Biosynthesis and uptake
Plants and most animals synthesize ascorbate from glucose. In primi-
tive fish, amphibians and reptiles, ascorbate synthesis takes place in kid-
ney, whereas liver is the site of synthesis in mammals. Humans, other
primates, guinea-pigs and a few species of fruit-eating bats cannot syn-
thesize ascorbate because the gene encoding L-gulonolactone oxidase
(GLO), the enzyme required for the last step in ascorbate synthesis, is
not functional [19]. Thus, dietary intake becomes vital, i.e. it is a vitamin.
The typical human diet contains both ascorbate and DHA; absorption
occurs in the enterocytes of the small intestine. While ascorbate is accu-
mulated in cells by Na+-dependent vitamin C transporters (SVCTs),
DHA is absorbed via a Na+-independent facilitative glucose transporters
(GLUTs) followed by intracellular reduction [20–24]. Under physiological
conditions, DHA concentrations in plasma are very low, 2 μM or less,
while plasma glucose levels are significantly higher (2–5 mM). Thus,
high intracellular ascorbate concentrations are mostly due to the uptake
of ascorbate by SVCT1 (SLC23A1) and SVCT2 (SLC23A2) [25,26]. Human
SVCT1 (Km ≈65–240 μM) resides largely in brush-border surfaces of in-
testinal and renal tubular cells to mediate absorption and re-absorption
[21]. Intestinal absorption and renal re-absorption determines the bio-
ð1Þ availability of ascorbate. Because of the limited affinity of SVCT1 for ascor-
bate, plasma ascorbate concentrations are tightly controlled [8]. SVCT2
(Km ≈8–62 μM) is found in a range of neural, neuroendocrine, exocrine,
and endothelial tissues as well as in osteoblasts [25,27]. Both isoforms of
SVCT are sensitive to the changes in ascorbate levels [28,29]; it has been
observed that high intracellular ascorbate lowered SVCTs in human plate-
lets and in an intestinal epithelial cell line Caco-2TC7, while low levels
up-regulated SVCTs. In addition, both SVCT1 and SVCT2 are remarkably
specific for L-ascorbate; ascorbate-2-O-phosphate, DHA, glucose, and
2-deoxyglucose are not transported by SVCTs. The SVCTs have a greater
 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457 445

Fig. 1. Structures of the chemical species associated with vitamin C. The structures in
blue are those forms that dominate the biochemistry of vitamin C.
affinity for L-ascorbate than D-isoascorbate; they depend on sodium to
create the electrochemical gradient across the plasma membrane, thereby
providing the energy required for uptake of ascorbate [27]. Recently an
ascorbate analog 6-bromo-6-deoxy-L-ascorbic acid has been identified
as completely specific for SVCTs but not GLUT1 or -3, the authors demon-
strated that 6-bromo-6-deoxy-L-ascorbic acid was taken up through
the SVCTs of human skin fibroblasts but not by neutrophils, which
accumulate DHA through GLUTs [30]. It has been hypothesized that the
SVCT pathway maintains intracellular levels of ascorbate under normal
physiologic conditions, whereas the GLUT-mediated DHA uptake is re-
stricted to specialized oxidizing conditions, such as those found in the sur-
rounding milieu of the activated neutrophils [30]. Given that cancer
development and progression is considered to have an inflammatory
component [31], it is highly possible that ascorbate, is oxidized in the ex-
tracellular environment, and then taken up by tumor as well as stromal
cells [32].
DHA is taken up with lower affinity (Km = 0.8 mM) than ascorbate
(Km = 0.2 mM), but the maximal rates of uptake by adult human
jejunum are similar for DHA and ascorbate (Vmax 28 vs. 35 pmol s−1
mg-protein−1) when glucose is absent [33]. Although the structure of
DHA has similarities to glucose, the influence of glucose on DHA uptake
varies between cell types. In adipocytes, neutrophils, osteoblasts and
smooth muscle cells, the uptake of DHA is largely inhibited by physio-
logical concentrations of glucose, which occurs to a lesser extent in
astrocytes, enterocytes, and renal tubular cells [27]. Mammalian facilita-
tive glucose transporters GLUT1 and GLUT3 mediate DHA transport
with a similar efficiency to that of glucose. GLUT4 transports DHA
with a higher affinity than 2-deoxyglucose (Km = 0.98 vs. 5.2 mM) but
a much lower Vmax [34]. Given that: (1) cellular uptake of ascorbate is
regulated by both glucose and insulin [35,36]; and (2) ascorbate infu-
sion has been shown to enhance glucose uptake and whole body glu-
cose disposal in healthy subjects and diabetics [37], it is clear that
more research is needed on the effects of ascorbate infusion and tissue
uptake.
Human erythrocytes express a high number of GLUT1, but have no
SVCT proteins [38]. The ascorbate level in erythrocytes is similar to the
plasma from which they were taken [39,40]. It is speculated that erythro-
cytes recycle and release ascorbate to help maintain plasma levels of
ascorbate. With the exception of erythrocytes, intracellular ascorbate con-
centrations are higher than extracellular fluids. Studies have shown that
ascorbate accumulates to millimolar concentrations in neutrophils, lym-
phocytes, monocytes, and platelets (Table 1). In circulating lymphocytes,
4 mM can be achieved in healthy young women with oral ascorbate
supplementation [41]. Intracellular ascorbate not only contributes to the
intracellular reducing environment, it can also quench extracellular oxi-
dants by transferring electrons across the plasma membrane [42]. In

Table 1
Ascorbate content of human tissues.

Tissue Ascorbate Ascorbate Ref.

(mmol/kg wet tissue) (mM)

Adrenal glands 1.7–2.3 [45]

Pituitary gland 2.3–2.8 [45]
Liver 0.8–1 [45]
Spleen 0.8–1 [45]
Pancreas 0.8–1 [45]
Kidneys 0.28–0.85 [45]
Skeletal muscle 0.17–0.23 [45]
Brain 0.74–0.85 [45,226,227,234]
Placenta 0.23–0.72 [48]
Plasma (healthy) 0.04–0.08 [41,45,51]
Red blood cell 0.04–0.06 [39]
Neutrophil 1.2 [41]
Lymphocyte 4.0 [41]
Monocyte 3.2 [41]
Platelet 3.7 [41,56]
Cerebral spinal fluid 0.15–0.25a [235–237]
Neuron 10 [238]
Glial cells 1 [238]
Lens 2.5–3.4 [46]
Corneal epithelium 12.5 [49]
Aqueous humor 0.4–1.1 [46]
Alveolar macrophage 0.32 [50]
Bronchoalveolar lavage 0.04–0.06 [51]
Saliva 0.04–0.05 [45]
a
Ascorbate levels in cerebral spinal fluid are approximately three to four times that of plasma.
446 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457

addition, there appears to be regulated ascorbate efflux from astrocytes to
neurons, thereby bolstering antioxidant defense [43]. Furthermore, the
release of ascorbate from cells may also reduce non-transferrin-bound
iron, thereby stimulating the uptake of iron by cells [44].
In human and animal tissues, the highest concentrations of ascorbate
are in the adrenal and pituitary glands (Table 1) [45–48]. Ascorbate is a
physiological reductant in the dopamine β-hydroxylase-catalyzed reac-
tion that converts dopamine to norepinephrine in the chromaffin
granules of adrenal medulla. Likewise, peptidyl glycine α-amidating
monooxygenase in the pituitary glands also requires high concentrations
of ascorbate to catalyze the amidation reaction of carboxyl-terminal gly-
cine residue of a number of peptides. In addition, millimolar levels of
ascorbate also found in eye tissues [46,49]; this may protect these tissues
from potential damage caused by solar radiation. Since tissues from
lungs are constantly exposed to relatively high concentrations of oxygen
and ROS, antioxidant enzymes and low-molecular-weight antioxidants,
such as ascorbate, serve to protect cells and tissues from oxidative dam-
age. Ascorbate levels in respiratory tract lining fluids are similar or lower
than those in plasma [50]. However, there appears to be increased ascor-
bate content in the epithelial lining fluid and alveolar macrophage of
smokers who have a relatively high dietary intake of vitamin C [51].
This increased content of ascorbate suggests that there is an adaptive de-
fense mechanism against the ROS derived from cigarette smoke.
Human cell culture experiments usually lack ascorbate, as these cells
are incapable of synthesizing this vitamin; in addition, there is no
source of vitamin C in typical cell culture media formulations. However,
cultured cells take up ascorbate, ascorbate-2-phosphate, as well as DHA
when supplemented in the media. DHA is rapidly transported into cells
by glucose transporters over a period of minutes while ascorbate and its
phosphate require SVCTs and can take several hours [52]. Ascorbate
2-phosphate is most likely hydrolyzed by membrane esterases with
the resulting ascorbate being transported into cells [53]. Compared to
the same concentration of ascorbate, cells accumulate intracellular
ascorbate via ascorbate 2-phosphate at a somewhat slower rate to
reach the same intracellular concentration [54]. However, ascorbate
2-phosphate does not oxidize in the media and thus does not produce
H2O2 as does ascorbate. Thus, the use of ascorbate 2-phosphate is pre-
ferred when studying the biology of vitamin C in cell culture. Millimolar
concentrations of ascorbate can be achieved intracellularly by
carrier-mediated mechanisms (Table 2). There is no detectable level
of DHA in cells as intracellular DHA is readily reduced by an array of bi-
ological reductants and enzyme systems [55,56].
At physiological conditions DHA rapidly hydrolyzes to 2,3-L-
diketogulonate (2,3-DKG), which is quite unstable [57]. 2,3-DKG subse-
quently decarboxylates yielding L-xylonate and L-lyxonate; these com-
pounds can enter the pentose phosphate pathways [58]. Alternatively,
L-erythrulose and oxalate can be produced from 2,3-DKG degradation. It
has been proposed that highly reactive L-erythrulose rapidly glycates
and crosslinks proteins. In fact, ascorbate-dependent modification of pro-
teins was proposed to occur in human lens during diabetic and
cataract formation. Oxalate monoalkylamide, one of the advanced
glycation end products (AGEs) is a major product induced by incubation
of human lens protein with DKG and other degradation product of ascor-
bate [59]. Oxalate is one of the major end products of ascorbate break-
down in humans, and it has the potential to crystallize as calcium
oxalate in the urinary space in susceptible people [60,61]. However, in a
study involving 16 cancer patients who had normal renal function, intra-
venous administration of ascorbate ranging from 0.2 to 1.5 g kg−1 body
weight, resulted in approximately 30 to 80 mg of oxalic acid being excret-
ed during the 6 h after the infusion [62]. While in primary hyperoxaluria,
oxalic acid excretion ranged between 100 mg d−1 and 400 mg d−1. Ox-
alate nephrocalcinosis and calcium oxalate stones develop over months to
years [63]. Overall, these studies suggest that patients with normal renal
functions and a time-limited course of ascorbate infusions would not
have an immediate risk of oxalate stone formation.
4. Biological functions
4.1. Ascorbate as enzyme cofactors of hydroxylases
In addition to the monooxygenases mentioned above, ascorbate is
involved in many physiological and biochemical processes involving

Table 2
Ascorbate in cultured cells.

Cell culture Treatment Intracellular ascorbate Ref

(mM)

A431 (human epidermoid carcinoma) 1 mM ascorbatea, 18 h 1 [239]

Adj.PC-5 mouse plasmacytoma 200 μM Ascb, 12 h 7 [240]
Alveolar macrophage (rat) 0.1 mM Asc, 30 min 3.2 [241]
Alveolar type II epithelial cells (rat) 0.1 mM Asc, 30 min 3.2 [241]
Astrocytes (rat) DHAc, 100 μM, 10 min 1.5 [248]
B lymphocytes 250 μM Asc, 4 h 0.7–1.5 [242]
EA.hy926 endothelial cells 0.3 mM Asc, 60 min 3–4 [244]
HepG2 hepatic epithelial cells 32 μM 14C-Asc, 3 min 1.1 [245]
HL-60 0.4–3 mM ascorbic phosphate 0.5 [246]
HUVEC 1 mM ascorbate, 18 h 3.5
INS-1 (rat pancreatic β-cell) 0.4 mM Asc, 16–18 h 2.4 [247]
K562 (human erythroleukemia) 500 μM DHA, 30 min 3 [248]
L6 skeletal myocytes (rat) 100 μM DHA, 10 min 12.5 [242]
d
MIA PaCa-2 pancreatic cancer 50 μM Asc, 4 h 6
1 mM Asc, 4 h 8
15 mM Asc, 4 h 18
Murine microvascular endothelial cells 0.5 mM Asc, 12 h 5 [249]
Osteoblasts (mouse) 1 mM Asc, 24 h 8
Primary hepatocytes 32 μM 14C-Asc, 3 min 0.41 [245]
SH-SY5Y neuroblastoma 2 mM Asc, 16–18 h 6 [250]
Skin fibroblasts 1 mM ascorbate, 18 h 0.4 [239]
U937 (human histocytic leukemia) 50 μM Asc or Asc-2-phosphate, 24 h 4 [54]
U937 1 mM Asc or Asc-2-phosphate, 24 h 7 [54]
a
Ascorbate.
b
L-Ascorbic acid.
c
Dehydroascorbate.
d
Thomas J van't Erve, personal communication.
 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457
enzymatic reactions that are catalyzed by members of the Fe 2+–
2-oxoglutarate-dependent family of dioxygenases. These dioxygen-
ases use 2-oxoglutarate as a co-substrate and as a source for two of
the four electrons needed for the reduction of O2; they transfer one
oxygen atom from O2 to the succinate product and one atom to the
protein substrate. Ascorbate is required for maintaining iron in the
ferrous state, thereby maintaining full activity of this class of en-
zymes. Reaction 3 [64,65].
3þ − 2þ
Fe  dioxygenase ðinactiveÞ þ AscH →Fe
•−
dioxygenase ðactiveÞ þ Asc :
Evidence that the Fe 2+–2-oxoglutarate-dependent dioxygenases
can modify proteins was gathered from the studies of hydroxylases
in collagen metabolism. Located within the lumen of the endoplasmic
reticulum, collagen prolyl-4-hydroxylase and prolyl-3-hydroxylase
are responsible for the insertion of the hydroxyl groups onto prolyl
residues, which is essential for the proper assembly of collagen [66].
There are at least three isozymes of collagen prolyl hydroxylase in
humans each with distinct α subunits; but they all have protein disul-
fide isomerases (PDIs) to serve as β subunits, which catalyze the for-
mation of intra- and inter-chain disulfide bonds during collagen
synthesis [67]. Collagens are major constituents of the extracellular
matrix (ECM), constituting ≈30% of total protein mass. Type I colla-
gen is the major structural protein in the interstitial ECM, while
type IV is prevalent in basement membrane [68]. Collagens in the ex-
tracellular matrix are important components of the physical barrier
against invasion and metastasis of cancer cells [88,89]. Ascorbate
has been shown to stimulate the production of types I and III collagen
in human skin fibroblasts [70]. Cameron and Pauling proposed that
mega doses of ascorbate would inhibit cancer growth by preventing
cancer cell invasion [71]. More recently, it has been shown that a
type IV collagen domain, produced as a recombinant protein, inhibits
the proliferation of capillary endothelial cells and blood vessel forma-
tion, resulting in the suppression of tumor growth [72]. However, it is
not known if very high levels of ascorbate will influence cancer
growth by mechanisms related to collagen synthesis.
4.2. Ascorbate as a cofactor in the HIF system
As novel members of the dioxygenase family, cytoplasmic prolyl
hydroxylases have been shown to regulate hypoxia-inducible tran-
scription factor (HIF) [73]. These enzymes have no PDI subunits but
have higher values of Km for oxygen than collagen prolyl hydroxy-
lases. HIF protein is composed of an oxygen-sensitive HIF-α subunit
and an oxygen-insensitive HIF-β subunit. In hypoxia, HIF-α is in-
duced, enters the nucleus, and forms a heterodimer with HIF-β,
which is constitutively expressed. In normoxia, HIF-α is efficiently
hydroxylated by prolyl hydroxylases. Hydroxylated HIF-α then
binds to the von Hippel–Lindau (pVHL) protein, which is part of an
ubiquitin ligase complex, leading to ubiquitination and proteasomal
degradation of the protein [74]. All three of the HIF prolyl hydroxy-
lases (PHD1, PHD2, and PHD3) recognize the C-terminal hydroxyl-
ation site with a higher affinity than the N-terminal hydroxylation
site [75]. In addition, an asparaginyl hydroxylase inhibits HIF through
the hydroxylation of a C-terminal asparagine residue, blocking
coactivator recruitment. Given that there are more than 800 direct
HIF target genes yielding more than 60 gene products that are regu-
lated by HIF [76–78], it is likely that the availability of ascorbate will
have a profound effect on many cell functions such as angiogenesis,
cell survival, glucose uptake, glycolysis and iron homeostasis.
As members of the Fe 2+–2-oxoglutarate-dependent dioxygenase
families, HIF prolyl- and asparaginyl hydroxylases require ascorbate
as a reducing agent to maintain iron in the ferrous state. Values of
Km for ascorbate with the HIF prolyl hydroxylases range from 140 to
447
300 μM [65], well below the millimolar concentrations of ascorbate
in most of cells in healthy individuals [55]. Supplementation of ascor-
bate in a lymphoma cell model in SCID mice has been shown to di-
minish HIF levels and the rate of tumor growth [79]. Lu et al.
demonstrated that the antitumorigenic effect of ascorbate is depen-
dent on the activity of HIF hydroxylases [80]. By analyzing endometri-
al tumor tissues for ascorbate and three of its targets GLUT-1, BNIP3
and VEGF, Kuiper et al. [81] found that HIF-1α and its targets were
all up-regulated in human tumor tissue. Tumors with higher ascor-
ð3Þ bate levels had low HIF-1 activation, while tumors with high HIF-1
activation and aggressive characteristics had lower ascorbate levels.
These important findings indicate that having high tissue levels of
ascorbate may protect against HIF-1 activation and aggressive
tumor behavior.
As a downstream factor produced upon activation of HIF, VEGF
plays an important role in tumor angiogenesis. In addition to direct
cytotoxicity effects, pharmacological ascorbate has been shown to
suppress capillary-like tube formation on cells grown on Matrigel
[82]. However, it is still unknown whether the suppression of angio-
genesis by pharmacological ascorbate is due to the production of
H2O2 [83] or through the inhibition of HIF [84].
4.3. Ascorbate as a cofactor in histone demethylation
The newly discovered histone demethylases containing Jumonji cat-
alytic domains, which can demethylate trimethylated lysines, also be-
long to the Fe 2+–2-oxoglutarate dioxygenase family [85]. As with
other members of this family, the Jumonji proteins have the ability to
bind Fe2+, use 2-oxoglutarate as substrate, and require ascorbate for
full catalytic activity. Furthermore, ascorbate supplementation also
leads to increased histone acetylation as well as increased expression
of HIF histone demethylases JMJD 1 and 2 in human embryonic stem
cells [86]. Thus, we can speculate that epigenetic status of tissue may
also depend on ascorbate to keep Fe2+–2-oxoglutarate dioxygenases
functional [87]. Indeed, Esteban et al. [88] also postulated that ascorbate
supplementation may enhance the function of epigenetic modifiers
during reprogramming of induced pluripotent stem cells (iPSCs), as
many of these modifiers are ascorbate-dependent dioxygenases. Con-
sidering that cancer cells have shared features of normal stem cells
[89] and DNA hypermethylation is associated with cancer development,
ascorbate and other nutritional supplements could prevent cancer by
modulating the components of the systems that set the epigenetic
code [90].
The necessity of ascorbate as a reducing agent to maintain full
functioning of an array of enzymes shows the importance of vitamin
C in maintaining the biochemical machinery of cells and tissues.
These functions are often overlooked as many researchers focus
only on the antioxidant abilities of ascorbate.
4.4. Ascorbate and cancer prevention
Epidemiologic evidence suggests that vitamin C-rich foods play a
protective role against development of cancer [91–94], plasma concen-
trations of ascorbate have been shown to be inversely associated with
cancer risk [95,96]; however, large scale randomized intervention
trials comparing antioxidants (vitamins A, C, E and β-carotene) supple-
ments given alone or in combination have not shown protective effects
[97–99]. One of the drawbacks of randomized control trials (RCT) for
nutrients is that most nutrients such as vitamins have threshold behav-
ior [100]. For example, scurvy only develops when serum ascorbate
levels are on the order of 8 μM or less [8]. The recommended dietary al-
lowance (RDA) for vitamin C is 75–125 mg daily; consistent intake of
60 mg d−1 will in general prevent scurvy for 30–45 days once vitamin
C intake ceased [101,102]. The fact that ascorbate is required to main-
tain full function of an array of enzymes indicates optimizing intake
448 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457
would optimize metabolism as well as prevent cancer and other degen-
erative disease [103,104].
5. Recycling of ascorbate
5.1. Reduction of ascorbate free radical
There are several enzymatic systems involved in the recycling of
ascorbate radical (Asc•−) and DHA. The reduction of ascorbate radical
by NADH-cytochrome b5 reductase has been shown in rat liver [105],
where most of the reductase activity is localized in the outer mitochon-
dria membrane. As a membrane-bound protein, NADH-cytochrome b5
reductase is also associated with the endoplasmic reticulum and the
plasma membrane [106]. Cytochrome b5 donates an electron to ascor-
bate radical, which in turn, draws electrons from NADH. On the other
hand, the transmembrane ascorbate radical reductase in erythrocytes
can use electrons from NADH or intracellular ascorbate to regenerate
extracellular ascorbate from ascorbate radical [107]. The plasma mem-
brane ascorbate radical reductase reduces ascorbate radical generated
in blood during oxidative stress as well as the ascorbate radical generat-
ed by reduction of α-tocoperoxyl radical during lipid oxidation. Reduc-
tion of ascorbate radical by thioredoxin reductase (TrxR) purified from
rat liver suggests that TrxR can also function as a cytosolic ascorbate
radical reductase, which may complement cellular ascorbate recycling.
Thus, there are several biochemical routes for the one-electron reduc-
tion of ascorbate radical to regenerate ascorbate.
5.2. Reduction of dehydroascorbate
DHA is unstable at physiological pH with a half-life is 5–15 min at
37 °C [108–110]. It is estimated that the concentration of DHA is less
than 2 μM in healthy humans; many reported measurements may
have overestimated DHA levels due to the ease of oxidation of
AscH − during sample processing. Extracellular DHA can be imported
into cells using glucose transporters present in cells, where DHA can
be reduced to ascorbate by glutathione (GSH) directly [111] or more
efficiently by glutaredoxin in the cytoplasm [112]. Purified rat liver
thioredoxin reductase also catalyzes NADPH-dependent reduction of
DHA [113]. Thus, electrons from NADPH can find their way to DHA
via thiols.
Purified bovine liver PDI has been found to react directly with DHA
and GSH to catalyze the reduction of DHA to ascorbate [112]. The
finding that PDI has DHA reductase activity is interesting since
human collagen prolyl 4-hydroxylase has PDI as a β subunit [69]. It
is possible that the β subunit also serves as DHA reductase to generate
ascorbate from DHA since collagen hydroxylases require ascorbate to
maintain iron in its ferrous state.
3α-Hydroxysteroid dehydrogenase belongs to the family of oxido-
reductases that catalyze NADPH-dependent oxidoreduction of a variety
of substrates. Del Bello et al. demonstrated that the NADPH-dependent
DHA reductase from rat liver cytosol is identical to 3α-hydroxysteroid
dehydrogenase [114]. The enzyme has a low affinity for DHA (Km =
4.6 mM) but a high affinity for NADPH (Km b 5 μM). Under pathological
conditions, decreases in GSH and increases in DHA could activate
3α-hydroxysteroid dehydrogenase.
Thus, DHA has many routes for its two-electron reduction to
regenerate ascorbate. Because of the relative instability of DHA, rapid
reduction to ascorbate also prevents loss of vitamin C. Ascorbate is in-
volved in a variety of biochemical functions that are fundamental to
the functioning of a wide variety of enzyme systems; thus efficient
recycling maintains the pool of ascorbate in cells and tissues.
The regeneration of ascorbate from ascorbate radical and DHA will
allow many cycles of ascorbate oxidation, thus one molecule of ascor-
bate can produce many molecules of H2O2. This mechanism is an im-
portant one when considering pharmacological ascorbate in cancer
treatment that will be discussed later.
6. Ascorbate as an antioxidant
The typical concentration of ascorbate in plasma from healthy humans
is about 40–80 μM (Table 1). At these levels ascorbate functions as an en-
dogenous antioxidant; for example, it serves as a co-antioxidant with vi-
tamin E to protect LDL from detectable oxidative damage induced by
aqueous peroxyl radicals [115]. Thermodynamically ascorbate readily do-
nates an electron to potentially damaging oxidizing radicals such as hy-
droxyl radical (HO•), alkoxyl radical (RO•), peroxyl radical (LOO•), thiol
radical (GS•), and tocopheroxyl radicals (TO•) [13]; this one-electron oxi-
dation of AscH− results in the production of the ascorbate radical (Asc•−).
Ascorbate radical is relatively unreactive, and can be reduced back to
ascorbate by NADH- and NADPH-dependent reductases [116]; Asc•−
can also undergo a pH-dependent disproportionation reaction, resulting
in the formation ascorbate and DHA, Reaction 1 [117,118].
Physiological concentrations of ascorbate have been shown to
inhibit LDL oxidation induced by endothelial cells, macrophages, copper
ions, and AAPH (2,2′-azo-bis (2-amidinoproprane) dihydrochloride)
[119,120]. An important feature of the action of ascorbate is its synergistic
interaction with vitamin E [121]. Vitamin E is lipid-soluble; it is a primary
antioxidant in LDL and lipid membrane oxidation [122]. Its one-electron
oxidation product, the α-tocoperoxyl radical, can be reduced by ascor-
bate. The two-electron oxidation product, tocopherol quinone, un-
dergoes ring breakage and thus vitamin E is lost [123]. In this context,
ascorbate is important for maintaining vitamin E and inhibiting lipid ox-
idation. It has been shown that doubling plasma ascorbate concentra-
tions by supplementation results in decreased rates of disappearance of
vitamin E in smokers [124]. This is the first in vivo demonstration that
ascorbate maintains vitamin E, most likely through “recycling”.
• •
LOO þ TOH→LOOH þ TO ð4Þ
− • •−
AscH þ TO →Asc þ TOH: ð5Þ
Ascorbate is clearly a co-antioxidant protecting lipid oxidation even in
iron-loaded human plasma [125]. In human plasma lipid peroxides
(cholesteryl ester hydroperoxides) were detectable only when serum
ascorbate was depleted; iron-induced lipid peroxidation was inhibited
by ascorbate in a concentration dependent manner [126]. Guinea pigs
with iron overload had greater oxidative lipid damage that is suppressed
with oral supplementation of ascorbate [127]. Thus, in the presence of cat-
alytic iron, robust nutritional levels of ascorbate protect from oxidative
damage.
7. Pro-oxidant effects of ascorbate
In the presence of catalytic metal ions, ascorbate can also exert
pro-oxidant effects [14,128,129]. Ascorbate is an excellent one-electron
reducing agent that can reduce ferric (Fe3+) to ferrous (Fe2+) iron,
while being oxidized to ascorbate radical (Reaction 6). Depending on co-
ordination environment, Fe2+ can readily react with O2, reducing it to su-
peroxide radical (Reaction 7), which in turn dismutes to H2O2 and O2
(Reaction 8).
− 3þ •− 2þ
AscH þ Fe →Asc þ Fe ð6Þ
2þ 3þ •−
Fe þ O2 →Fe þ O2 ð7Þ
•− •− þ
O2 þ O2 þ 2H →H2 O2 þ O2 : ð8Þ
In a classic Fenton reaction, Fe2+ reacts with H2O2 to generate Fe 3+
and the very oxidizing hydroxyl radical, Reaction 9. The presence of
ascorbate can allow the recycling of Fe3+ back to Fe2+, which in turn
will catalyze the formation of highly reactive oxidants from H2O2.
2þ 3þ •
Fe þ H2 O2 →Fe þ HO ðFenton reactionÞ: ð9Þ
 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457
Depending on concentrations, the effects of ascorbate on models
of lipid peroxidation can be pro- or antioxidant [14,130]. There is con-
siderable variability in the literature; this variability appears to be a
result of the different concentrations and form of transition metal
ions in the experiments and the media [131].
The prooxidant effects of ascorbate may be important in vivo
depending on the availability of catalytic metal ions. In healthy individ-
uals, iron is largely sequestered by iron binding proteins such as trans-
ferrin and ferritin [132,133]. Transferrin is a glycoprotein that is
synthesized in the liver. It is the major circulating iron binding protein
with a high affinity, but low capacity for iron; this iron is essentially
redox inactive [134]. Transferrin avidly binds to the transferrin receptor
on the cell surface. The transferrin–transferrin receptor complex is in-
ternalized to an endosome, which releases iron in acidic conditions.
The ferric iron released is reduced and transported to the cytoplasm
where it is either utilized for synthesis of iron-containing proteins or
bound in ferritin, an iron storage protein. Ferritin is capable of seques-
tering up to 4500 atoms of iron, but is normally only 20% saturated.
Iron stored in ferritin can be released by appropriate reductants in the
presence of a chelator or by degradation of ferritin in the lysosome.
Iron can be released from ferritin by biological reductants such as
thiols, ascorbate and reduced flavins [135]. The released iron enables
cells to synthesize cytochromes and iron-containing enzymes. How-
ever, uncontrolled release of iron from ferritin has the potential to
form HO •, which can damage critical cellular components [136–138].
In pathological situations, such as thalassaemia or hemotochro-
matosis, non-transferrin-bound iron is present. Thus, supplemental
ascorbate without administration of an iron chelator can lead to delete-
rious effects [14]. Tissue damage resulting from ischemia/reperfusion is
another example of increased availability of catalytic metal occurring in
vivo [139]. Intravenous ascorbate prior to vascular surgery increased
concentrations of ascorbate radical and lipid hydroperoxides suggesting
that catalytic iron released into the circulation during the ischemic
phase of the surgery with ascorbate may promote iron-induced lipid
peroxidation [140,141].
Elevated levels of catalytic metal ions have also been demonstrat-
ed in chronic inflammatory diseases [142]. There is an increased de-
position of iron proteins in the synovial membranes in rheumatoid
arthritis. Ascorbate radical has been detected in synovial fluid from
patients with synovitis disease indicating that catalytic iron is in
part responsible for the decreased levels of ascorbate and increased
levels of DHA [141]. In addition, ascorbate concentrations were de-
creased while levels of catalytic iron increased in patients with sepsis,
compared to healthy subjects [143].
Although there is no direct evidence that catalytic iron is increased
in tumors, many patients with malignant disease have elevated
serum or tissue ferritin concentrations [144–146]. Raised levels of cir-
culating ferritin are found in childhood Hodgkins lymphoma and are
associated with poor survival [147]; serum ferritin levels have been
shown to be related to the stage of the disease and tumor volume in
cervical cancer [148]. Furthermore, studies by Feng et al. [149] have
shown that the peritoneal and subcutaneous microvessels of normal
mice were largely impermeable to circulating ferritin, but in mice
bearing solid or ascites tumors, circulating ferritin was found in the
basal lamina and extravascular space, suggesting that tumor vessels
are hyperpermeable to circulating macromolecules such as ferritin.
In fact, ferritin staining was detected in stroma and histiocytes sur-
rounding neoplastic cells of breast carcinoma tissue of patients
[150]. Extracellular metal-containing proteins have been proposed
to be essential for the pro-oxidant effects of ascorbate [10,129],
iron-saturated ferritin could be a potential candidate as source of cat-
alytic iron. A recent study by Deubzer et al. [151] demonstrated that
ferritin released by neuroblastoma cells enhanced pharmacologic
ascorbate induced-cytotoxicity, indicating that ferritin with high
iron-saturation could be a source of catalytic iron [152]. Consistent
with this, ascorbate has also been shown to be capable of releasing
449
iron from cellular ferritin [158]. Ferritin is only one candidate as a
source of catalytic iron; extracellular iron chelates are present in tis-
sue and seem to be increased under pathological conditions [159]. It
is clear that only low levels of catalytic metals are needed to substan-
tially increase the rate of oxidation of ascorbate [15,18]. These many
observations provide insights on the mechanism by which pharmaco-
logic concentrations of ascorbate have potential in treating certain
types of cancer.
8. Ascorbate and cancer treatment
The use of high-dose ascorbate in treating cancer patients began in
the 1970s. These early studies demonstrated beneficial effects of
high-dose ascorbate [155–158]. However, two double-blinded, ran-
domized clinical trials at the Mayo Clinic did not show any benefit
[169,160]. Subsequently, use of ascorbate for cancer treatment was
considered ineffective and dismissed by the research and medical
communities. However, a marked difference existed in these studies.
Cameron's group gave patients ascorbate intravenously as well as
orally, while patients in the Mayo Clinic trials received only oral
ascorbate. Some years later, clinical data were generated that demon-
strated that when ascorbate is given orally, plasma concentrations are
tightly controlled [101]. At oral doses of 200 mg, the steady-state
plasma concentrations are ≈80 μM. As doses exceed 200 mg, relative
absorption decreases, urine excretion increases and the fraction of
bioavailable ascorbate is reduced [7]. Peak plasma values do not ex-
ceed ≈220 μM even after maximum oral dose of 3 g 6 times daily
[8]. In contrast, when ascorbate is administered intravenously, milli-
molar concentrations can be achieved. In fact, infusion of 10 g of
ascorbate in cancer patients results in plasma concentrations of 1 to
5 mM [161,162]. Thus, only intravenous administration of ascorbate
can yield high plasma levels, i.e. pharmacological levels.
8.1. The role of hydrogen peroxide generation and removal
In vitro, the toxicity of ascorbate centers on the generation of H2O2
by ascorbate upon its oxidation [9,10,12,163,164]. The true autoxida-
tion of ascorbate, i.e. in the absence of catalytic metals, via reaction 2
will generate considerable amounts of H2O2 when ascorbate is at mil-
limolar levels. For example, in an aqueous solution containing 20 mM
ascorbate at pH 7.4, the concentration of ascorbate dianion, Asc 2−,
will be on the order of 1 μM. This will result in a flux of H2O2 on the
order of 10 nM s −1, in a typical cell culture experiment.
Catalytic iron (and perhaps copper) in the media and serum will
increase the rate of ascorbate oxidation and associated generation of
H2O2. Clement et al. found that Dulbecco's modification of Eagle's
MEM (DMEM) generates more H2O2 than RPMI 1640 during a
6-hour incubation with increasing concentrations of ascorbate [165].
In addition to adventitious iron, DMEM has 0.25 μM Fe (NO3)3 in its
formulation, which could contribute to this greater production of
H2O2 in DMEM than that from RPMI. Serum usually contains total
iron at a range of 10 to 50 μM, which could also be another factor
that causes the variable toxicity of ascorbate in different media; how-
ever, some of this iron will be redox inactive as it will be sequestered
in transferrin [134]; there will undoubtedly be a highly variable
amount of iron present that has nonspecific peroxidase activity
[166]. This iron will actively catalyze the oxidation of ascorbate.
Thus, the amount of catalytic iron present in typical cell culture
media is highly variable. Even with careful attention to detail, this
can lead to considerable variability in experimental results, even in
the same set of experiments.
α-Keto acids such as pyruvate and α-ketoglutarate directly react
with H2O2 when present in cell culture media [172];
CH3 ð¼ OÞð¼ OÞOH þ H2 O2 →CH3 Cð¼ OÞOH þ CO2 ð10Þ
450 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457
this will result in an apparent lowering of the toxicity of ascorbate
[168]. Thus, the presence of α-keto acids in media must be taken
into consideration in the design and interpretation of data from ex-
periments where the oxidation of ascorbate and associated produc-
tion of H2O2 are central to the study.
Because cells rapidly and efficiently remove extracellular H2O2, ob-
served toxicities or cell killing induced by ascorbate is an inverse func-
tion of cell density [151,153,169]. There is an array of intracellular
antioxidant enzymes involved in the removal of H2O2, such as catalase,
glutathione peroxidase, and the peroxiredoxins. Although modest cata-
lase activity has been detected in heart mitochondria [170], catalase is
predominantly located in peroxisomes of mammalian cells. Concomi-
tant administration of the catalase inhibitor amino-1,2,4-triazole has
been shown to enhance ascorbate toxicity to Ehrlich ascites cells in
vitro [171]. Using adenovirus constructs containing human catalase
cDNA, Du et al. [12] demonstrated that overexpressing intracellular cat-
alase protected cells from ascorbate-induced cytotoxicity. These results
provide evidence supporting a fundamental role for H2O2 in
ascorbate-induced toxicity.
Cytosolic glutathione peroxidase (GPx1), another antioxidant en-
zyme that removes peroxide, is widely distributed in tissues. GPx1
catalyzes GSH-dependent reduction of H2O2 to water. Gaetani et al.
[172,173] have shown that the intracellular concentration of H2O2
in human erythrocytes is inversely proportional to the activity of cat-
alase and GPx-1. GPx1 is responsible for eliminating low concentra-
tions of H2O2; however, at high fluxes of H2O2 recycling of GPx1 by
GSH becomes rate-limiting [174]; at high fluxes of H2O2 catalase is
the enzyme that is principally responsible for removal of H2O2 [175].
Another antioxidant system that contributes to removal of H2O2
within cells is the family of peroxiredoxins, in particular, peroxiredoxin
2 (Prx2). Prx2 is the third most abundant protein in erythrocytes
[176]. The rate constant of human Prx2 reacting with H2O2 is
1.3 × 10 7 M − 1 s − 1 [177] which is similar to that of catalase (1.7 ×
10 7 M − 1 s − 1 [178]). These complementary enzymes systems for
the removal of intracellular H2O2 in RBCs will also make these cells
efficient sinks for H2O2.
8.2. Ascorbate-induced cytotoxicity in vitro
Chen et al. [9,11] have demonstrated that some cancer cells have in-
creased sensitivity to ascorbate-induced cytotoxicity compared to nor-
mal cells. In a complementary study, Du et al. [12] demonstrated that
pancreatic cancer cells are more sensitive to pharmacological concen-
trations of ascorbate than their normal cell counterparts. The difference
in sensitivity between normal and cancer cells towards ascorbate may
be due to low levels of antioxidant enzymes and high endogenous levels
of ROS in cancer cells [179–181]. The relative lower activities of catalase,
glutathione peroxidase, and peroxiredoxins in cancer cells could poten-
tially contribute to less efficient removal of H2O2 and increased sensitiv-
ity to ascorbate-induced cytotoxicity.
The rate of oxidation of ascorbate is typically a function of the level
of catalytically active iron and copper in solution. Iron in cell culture
media contributes significantly to the rate of H2O2 generation.
Deferoxamine (Desferal® or DFO) is an iron-chelating agent that ren-
ders iron catalytically inactive with respect to ascorbate oxidation
[15]. Although hydrophilic, DFO is cell-permeable; short-term expo-
sure to DFO is not effective in accessing cytosolic labile iron pool
(LIP) of cells from different origins [182]. However, short-term
pre-incubation of cells to DFO appeared to protect cells from
ascorbate-induced toxicity [184]. These observations indicate that
DFO either is effective in accessing endosomal iron [182] or iron asso-
ciated with cellular membranes [183]. Other cell culture studies have
demonstrated that Fe 2+ in the media can actually protect cells from
the oxidative damage resulting from exposure to extracellular H2O2
[184,185]. Although hydroxyl radicals are produced via the Fenton re-
action, most will not induce cell damage, but rather disappear by
reactions with components of the media. The key is that H2O2 is re-
moved. Thus, interpretation of data from experiments with iron,
ascorbate, H2O2 or some combination is not always straightforward.
Oxidative stress has been shown to increase the levels of catalytic
iron in tissues. A series of electron paramagnetic resonance (EPR)
studies demonstrated that ultraviolet light increased the levels of la-
bile iron in skin [186]; heat stress-induced oxidative events increased
the level of labile iron in liver [187]; and ischemia reperfusion in-
creased the level of catalytic iron in the heart [188]. In addition, ion-
izing radiation and some chemotherapeutic drugs have been shown
to increase catalytic free iron levels [189–191]. Since it takes only
very low concentrations of catalytic metals to bring about the rapid
oxidation of ascorbate [192,193], approaches that increase catalytic
iron could potentially enhance the cytotoxicity of pharmacologic
ascorbate in vivo [194].
Porphyrin-based SOD mimics have a redox-active metal (Mn, Fe,
and Cu) center and a stable porphyrin complex. The dismutation of
O2•− by Mn porphyrin complexes involves two steps in which the Mn
center cycles between Mn(III) and Mn(II): the first step is the reduc-
tion of Mn(III) by O2•− to yield Mn(II) and O2 and the second step the
oxidation of Mn(II) by O2•− to yield H2O2 and return the manganese
to its resting state as Mn(III) porphyrin [195]. However, in the pres-
ence of a reductant such as ascorbate, Mn porphyrins function as su-
peroxide reductases rather than dismutases; Mn(III) can be reduced
to Mn(II) by ascorbate while Mn(II) can react with O2 forming O2•−,
which subsequently forms H2O2 and O2, Reaction 8. The prooxidant ef-
fects of Mn porphyrin and ascorbate have been studied by several
groups. Gardner et al. [196] have demonstrated that MnTM-4-PyP 5+
catalyzes the oxidation of ascorbate in vitro. Zhong et al. [197] have
shown synergistic killing of human prostate cancer cell RWPE-2 by
MnTM-4-PyP 5+ and ascorbate. Most recently, Tian et al. [198] have
shown that MnTM-4-PyP 5+ synergizes with ascorbate inhibiting the
growth of prostatic, pancreatic, and hepatic cancer cells. Furthermore,
the cytotoxic effects of two Mn(III) alkylpyridylporphyrins (MnTE-2-
PyP 5+ and MnTnHex-2-PyP 5+) and ascorbate have been demonstrat-
ed in Caco-2, HeLa, HCT116, and 4T1 cells [199,200]. The more lipo-
philic MnTnHex-2-PyP 5+ appeared much more effective. Given that
several Mn porphyrins have already been tested in vivo as SOD mi-
metics, and by themselves have shown low toxicities at micromolar
levels [195], there is great potential for using Mn porphyrins as an ad-
juvant to enhance the efficacy of pharmacologic ascorbate.
8.3. High dose ascorbate in animal studies
As mentioned above, the uptake of oral ascorbate in humans is
tightly controlled by the gut and kidney filtration [7]. Rats receiving
ascorbate by gavage (0.5 mg g −1) increased blood and extracellular
concentration to peak levels b 150 μM [10]. By contrast, concentra-
tions reached peak levels of nearly 3 mM in rats receiving intraperito-
neal injections, while intravenous injection increased peak levels to
>8 mM. In a similar study, mice receiving bolus intravenous injec-
tions of ascorbate (1 g kg −1), resulted in plasma concentrations of
15 mM [164]. Supplementation of ascorbate in drinking water at
1 g kg −1 only increased plasma concentrations to b 50 μM. These re-
sults clearly indicate that pharmacologic concentrations of ascorbate
cannot be obtained by oral administration.
In addition to the finding that millimolar levels of ascorbate were
achieved by parenteral administration, Chen and colleagues [10]
demonstrated that ascorbate radical was formed in extracellular
fluid but was not detectable in whole blood. Moreover, H2O2 was
detected in extracellular fluid, but not in the blood; H2O2 correlated
with ascorbate radical concentration. These results indicate that plas-
ma membrane associated ascorbate radical reductase and high levels
of catalase, glutathione peroxidase, and peroxiredoxin enable eryth-
rocytes to act as a sink for extracellular ascorbate radical and H2O2
[176,107]. On the other hand, ascorbate could be oxidized by catalytic
 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457 451

iron associated with damaged proteins in the extracellular space
[201–203, 209]. Considering that the permeability of tumor vessels is
much higher compared to normal endothelium, tumor endothelium
may permit the outflow of macromolecules, such as albumin, to intersti-
tial fluid [169,204]. The fluid that accumulated in the peritoneal
cavities of ascites tumor-bearing mice had a protein concentration
several-fold greater than that of normal peritoneal fluid, with approxi-
mately 85% of the protein level of plasma, including albumin, in a pro-
portion similar to that found in plasma [205,206]. Human albumin is
the most abundant protein in the plasma; and has the capacity to bind
to metal ions such as Cu2+ [207]. The first four amino acids of the
N-terminus of human albumin Asp-Ala-His-Lys forms a tight-binding
site for Cu2+ [207]. In fact, almost thirty years ago, Linus Pauling and
his colleagues [194] designed a copper:glycylglycylhistidine complex
that mimics the binding of albumin to copper; it enhanced the antitumor
activity of ascorbate against Ehrlich ascites tumor cells. In addition, ex-
tracellular fluids contain little or no catalase activity, and low levels of
SOD and GPx [208]. Thus, the H2O2 generated by ascorbate oxidation in
the extracellular space could accumulate to a concentration greater
than the intracellular level resulting in net rate of diffusion across the
cell membrane into cells, resulting in toxicity to cancer cells [10].
Pharmacological ascorbate inhibits tumor growth in mice. In mice
bearing tumor xenografts of pancreatic cancer, treatment with
4 g kg −1 ascorbate (i.p., twice daily) significantly decreased the rate
of tumor growth [11,12,164] (Table 4). Upon cessation of treatment
with pharmacological ascorbate, the rate of tumor growth increased.
This rate was similar to the rate observed in the control group, i.e. no
ascorbate (Du et al., unpublished results). In this tumor model phar-
macologic ascorbate as a single agent was cytostatic, not cytotoxic. Ob-
viously, the use of intravenous high dose ascorbate as a single agent for
cancer was not curative. To test the combination effect of standard che-
motherapy with ascorbate, Espey et al. [209] have shown that synergis-
tic cytotoxic effects can be achieved with gemcitabine and ascorbate in
pancreatic cancer both in vitro and in a nude-mouse model. These data
provide support for investigating the use of pharmacologic ascorbate as
an adjuvant for conventional cancer chemotherapies.

Table 3
Pro-oxidant effects of ascorbate in cultured cells.

Cell line Treatment Effects Ref

Primary human diploid fibroblasts GM5399 20–500 μM Asc Inhibit growth, DNA damage [251]
Normal human skin fibroblast CCD-25 SK Sodium ascorbate, 0.56–2.8 mM No effect [210]
Normal human colon fibroblast CCD-18 Co Sodium ascorbate, 0.1–0.4 mM No effect [210]
Human embryonic fibroblast Sodium ascorbate, 0.2 mM Inhibit growth [252]
HRE human normal renal epithelial cells Asc 1.2 mM Promote growth [253]
Human normal myeloid cells L-Ascorbic acid, 0.3 mM No effect [254]
Primary chicken embryo fibroblast Sodium ascorbate, 0.2 mM Inhibit growth [252]
AN3 CA endometrial adenocarcinoma Sodium ascorbate, 0.56–2.8 mM Inhibit growth [210]
Ehrlich ascites carcinoma Ascorbate + 3-amino-triazole Synergistic killing [171]
5637 human bladder cancer Asc EC50 b 5 mM Inhibit growth [11]
T24 human bladder cancer Asc + vitK3 Synergistic, [255]
Autoschizis,
Necrosis, apoptosis
MB231 human breast cancer 5 mM Asc Decrease clonogenic survival [9]
MCF-7 human breast cancer 5 mM Asc Decrease clonogenic survival [9]
MCF-7 human breast cancer Asc + porphyrin Apoptosis, cell cycle disruption [256]
Hs587t human breast cancer 5 mM Asc Decrease clonogenic survival [9]
HeLa human cervical cancer Sodium ascorbate, 0.25 mM; No effect [11,257]
Asc EC50 > 5 mM
HeLa human cervical cancer Asc + MGd (Motexafin gadolinium) Apoptosis, [257]
Lysosomal rupture
Normal human colon fibroblast CCD-18-Co Sodium ascorbate, 0.1–0.8 mM No effect [210]
Human colon carcinoma cells Sodium ascorbate, 0.1–3.2 mM Inhibit growth [210]
HEp-2 human epidermoid larynx carcinoma Asc 500 μM + 25 μM vitB12b Apoptosis, DNA damage [258]
A549 human lung cancer Asc EC50 b 5 mM Inhibit growth [11]
HepG2 human hepatocellular carcinoma Asc, Asc + MnTMPyP Decrease clonogenic survival, mitochondria damage. [198]
DU 145 human prostate cancer cells Sodium ascorbate, 1–10 mM; Inhibit growth [198]
Asc + MnTMPyP
LNCaP human prostate cancer cells Sodium ascorbate, 1–10 mM; Inhibit growth [198]
Asc + MnTMPyP
PC-3 human prostate cancer Sodium ascorbate, 0.56–2.3 mM; Inhibit growth [198]
Asc + MnTMPyP
MIA PaCa-2 human pancreatic carcinoma Sodium ascorbate, 0.56–2.8 mM; Inhibit growth [210]
MIA PaCa-2 human pancreatic carcinoma Asc 1–5 mM Cytotoxic, autophagy [12]
PANC-1 Asc, Asc + MnTMPyP Decrease clonogenic survival [198]
Pan02 mouse pancreatic cancer Asc 2.5–10 mM cytotoxic [11]
Human acute leukemic cells L-Ascorbic acid, 0.3 mM Inhibit growth [254]
U937 human histocytic leukemia cells L-Ascorbic acid, 50–300 μM Inhibit growth [163]
Human chronic lymphocytic leukemia Asc 0.3–5 mM; Cytotoxic [259]
Asc + ATO (Arsenic trioxide)
Kelly human neuroblastoma 0.6–5 mM Cytotoxic [151]
SK-N-SH human neuroblastoma 0.6–5 mM Cytotoxic [151]
Mouse neuroblastoma Sodium ascorbate + 5-FU Inhibit growth [222]
Sodium ascorbate + X-irradiation
9 L rat glioblastoma Asc 2.5–10 mM Cytotoxic [11]
LN18 human glioblastoma cell lines, Asc + γ-irradiation Double-stranded DNA breaks, G2/M arrest block [224]
human primary glioblastoma cells,
GL261 mouse astrocytoma cell line
B16F10 murine melanoma Asc 0.05–0.2 mM Cell cycle arrest [260]
Chinese hamster ovary (CHO) cells Ascorbate + misonidazole Inhibit growth [261]
452 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457

8.4. Clinical studies
Although the two clinical trials using oral ascorbate at the Mayo
Clinic failed to show any benefit, numerous case reports have provided
encouraging results with intravenous ascorbate therapy [162,210,211].
However, most of the cases were reported without sufficient detail
or follow-up for evaluation. Padayatty et al. [212] examined three
well-documented cases in accordance with National Cancer Institute
(NCI) Best Case Series guidelines. These cases suggest that high-dose,
intravenous ascorbate therapy prolonged the survival of the patients
with advanced cancer.
Intravenous ascorbate therapy is likely to be safe in most patients ex-
cept under certain conditions [213]. Because the removal of H2O2 gener-
ated in blood requires glutathione, which in turn requires NADPH
generated by G6PD, the rate-limiting enzyme of the pentose phosphate
pathway [214], high-dose ascorbate can induce intravascular hemolysis
in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency
[215–217]. One end-product of ascorbate oxidation is oxalic acid, which
has the potential to crystallize as calcium oxalate in the urinary space in
patients with pre-existing renal insufficiency [218]. In addition, rare
cases of massive tumor hemorrhage have been reported in patients
with advanced cancer following high-dose intravenous ascorbate
[155,219]. Thus, as always, caution is needed, but there are clear indica-
tions for a potential role for ascorbate in cancer treatment.
Three phase I clinical trials of intravenous ascorbate in patients
with advanced cancer measured plasma concentrations of ascorbate
and evaluated clinical consequences [162,220,221]. In the study by
Riordan and colleagues, patients were given continuous infusion of
0.15 to 0.7 g kg −1 day −1 for up to eight weeks; in the Hoffer study,
ascorbate was administrated three times per week at fixed doses of
0.4, 0.6, 0.9 and 1.5 g kg −1; and in the most recent study by Monti
et al. [221], patients received 50, 75 and 100 g per infusion (three in-
fusions per week) for 8 weeks with concomitant i.v. gemcitabine and
oral erlotinib. In the Riordan study, ascorbate concentrations in serum
during therapy ranged from 0.28 to 3.8 mM. In the Hoffer study, peak
plasma ascorbate concentrations ranged from 2.4 to 26 mM. When
1.5 g kg −1 was administered, plasma ascorbate concentrations
exceeded 10 mM for ≈ 4.5 h [220], levels that have been shown to in-
duce cell killing in a variety of cancer cells [9,11,12,164]. Finally in the
Monti study, patients that received 100 g of ascorbate achieved peak
plasma concentrations between 25 and 32 mM. All three trials
demonstrated that high-dose intravenous ascorbate was well tolerat-
ed in cancer patients with normal renal function; the combination of
ascorbate infusion with standard of care chemotherapies did not in-
crease toxicity.
8.5. Ascorbate and conventional cancer therapy
The cytotoxicity of high-dose intravenous ascorbate is dependent
on the ascorbate-induced production of H2O2. Hydrogen peroxide
can deplete intracellular GSH, causing oxidative stress. In high doses
ascorbate acts as a pro-oxidant to kill cancer cells. Initial concerns
that ascorbate might reduce the effectiveness of standard chemother-
apy and/or radiation therapy have not been demonstrated. Numerous
reports suggest that pharmacological ascorbate may actually increase
the efficacy of several chemotherapeutic drugs and radiation in vitro
[164,222–224] (Table 3). Espey et al. [209] demonstrated that phar-
macologic concentrations of ascorbate with gemcitabine resulted in
a synergistic cytotoxic response in pancreatic tumor cell lines;
gemcitabine–ascorbate combinations administrated to mice bearing
pancreatic tumor xenografts consistently enhanced inhibition of
growth compared to gemcitabine alone. Case reports [161,212] indi-
cate that intravenous ascorbate can be safely used with chemothera-
py and radiotherapy and could potentially enhance the effects of
conventional cancer therapy. Furthermore, intravenous ascorbate
has been shown to improve the quality of life in breast cancer pa-
tients during chemo-/radiotherapy and aftercare [225]. Clinical trials
using combinations of chemotherapy and intravenous ascorbate are
underway at several institutions (www.clinicaltrials.gov).
9. Perspectives
The inhibition effects of pharmacologic ascorbate on tumor growth
have been confirmed in many laboratories (Table 4). In murine models
elevated ascorbate levels in plasma were verified following i.p. adminis-
tration [11,164]. It is still unknown whether cells and tissues levels are
also elevated [226,227]. Human platelets supplemented with 500 μM
Asc for 30 min showed a decrease in SVCT2 levels, while in Asc depleted
platelets SVCT2 expression was higher than native ones [29]. Thus in
vivo studies for possible changes in SVCT expression levels following
ascorbate infusion are needed. Another issue is a possible transient
withdrawal effect upon cessation of treatment with pharmacologic

Table 4
Pro-oxidant effects of ascorbate in animal models.

Species Cell type Treatment Effects Ref

Balb/c nude mice HT29 colon cancer Asc 15 mg, 100, 1000 mg/kg, i.p., daily. 4 weeks. 7/7 mice survived at 1000 mg/kg. No carcinogenic [262]
invasion. Tumor volumes decrease. ARSs and EiFs
genes down regulated.
Balb/c nude mice K562 leukemia Vit K3 10 mg/kg, i.p.; Asc + vit K3 decrease tumor growth. [263]
Asc 1 g/kg, i.p.
Ncr nude mice Ovcar5 ovarian cancer Asc 4 g/kg, i.p., twice daily. 30 days. No adverse effects. Decrease tumor growth. [11]
Ncr nude mice Pan02 pancreatic cancer Asc 4 g/kg, i.p., daily. 14 days. No adverse effects. Decrease tumor growth. [11]
Ncr nude mice 9 L rat glioblastoma Asc 4 g/kg, i.p., twice daily. 12 days. No adverse effects. Decrease tumor growth. [11]
Prevent metastases.
Ncr nude mice Pan02 pancreatic cancer Asc 4 g/kg, i.p., daily; No side effects except osmotic stress. [209]
Gemcitabine 30, 60 mg/kg, i.p., every 4 days. 21 days. Asc + Gem significantly
decrease tumor growth.
Ncr nude mice PANC-1 pancreatic cancer Asc 4 g/kg, i.p., daily; No side effects except osmotic stress. [209]
Gemcitabine 30, 60 mg/kg, every 4 days. 33 days. Asc + Gem significantly
decrease tumor growth.
Nude mice MIA PaCa-2 pancreatic cancer Asc 4 g/kg, i.p., twice daily. 14 days. No adverse effects. Decrease tumor growth. [12]
NMRI mice TLT Murine hepatoma Asc 1 g/kg, i.p., daily. 30 days. Decrease tumor growth. [164]
Nude mice H322 non-small cell lung cancer Asc 250 mg/kg, i.p.; Asc synergistic with 101 F6 decrease tumor growth. [264]
101 F6 nanoparticle i.v.
SCID mice EHMS-10 mesothelioma cells Asc 0.88, 8.8 g/mouse, i.v. single dose. Decrease tumor growth. [265]
Balb/c mice Murine sarcoma S180 cells Asc 5.5, 30 mg/mouse, i.p., every two days. Decrease tumor growth; inhibit bFGF, VEGF [266]
and MMP2 genes.
Balb/c mice Murine sarcoma S180 cells Asc 1.5 mg/g, i.p., every three days. Inhibit tumor establishment; RKIP and annexin [267]
A5 levels increase.
 J. Du et al. / Biochimica et Biophysica Acta 1826 (2012) 443–457
ascorbate [228]. Male guinea pigs that received ascorbate 1 g kg−1
body weight per day by i.p. for 4 weeks had elevated plasma and uri-
nary levels; however, in the weeks after the treatment withdrawn,
mean plasma and urinary ascorbate were significantly lower than nor-
mal [229]. It is unknown if a similar rebound effect would occur in
human subjects following high doses i.v. ascorbate. F2-isoprostane is a
biomarker for lipid peroxidation in vivo, and its quantification in plasma
and urine has emerged as the most reliable method to assess systemic
oxidative stress in humans and animals [230–232]. For healthy young
women taking oral doses of ascorbate 30–2500 mg daily, plasma and
urine F2-isoprostanes were unchanged [41]. It is unclear whether
plasma F2-isoprostanes will change following pharmacologic ascorbate
infusion in cancer patients. Furthermore, since pharmacological ascor-
bate is a prodrug for H2O2 generation, there should be many applica-
tions where delivery of H2O2 via pharmacological ascorbate may have
clinical benefit, such as infectious diseases caused by viruses, bacteria,
and other pathogens [10,233].
10. Summary
More than eighty years since its discovery, our understanding of
the functions of ascorbic acid has evolved from the prevention of
scurvy to its potential use as a therapeutic drug for cancer treatment.
Ascorbate maintains Fe 2+ of collagen hydroxylases in an active state;
therefore it plays a pivotal role in collagen synthesis; parallel reac-
tions with a variety of dioxygenases affect the expression of a wide
array of genes, for example via the HIF system, and possibly the epige-
netic landscape of cells and tissues. The ability to donate one or two
electrons makes ascorbate an excellent reducing agent and antioxi-
dant. However, in the presence of catalytic metals, ascorbate also
has pro-oxidant effects, where the redox-active metal is reduced by
ascorbate and then in turn reacts with oxygen, producing superoxide
that subsequently dismutes to produce H2O2.
Apart from its biochemical functions that are met by healthy nutri-
tional levels, recent pharmacokinetic data indicate that intravenous ad-
ministration of ascorbate bypasses the tight control of the gut and renal
excretion; thus, intravenous administration of ascorbate will produce
highly elevated plasma levels; this ascorbate will autoxidize resulting
in a high flux of extracellular H2O2. This H2O2 will readily diffuse into
cells challenging the intracellular peroxide-removal system, initiating
oxidative cascades. These high fluxes of H2O2 appear to have little effect
on normal cells but can be detrimental to certain tumor cells. Knowl-
edge and understanding of these mechanisms brings a rationale to the
use of high-dose ascorbate to treat disease and thereby is reviving inter-
est in the use of i.v. ascorbate in cancer treatment. The full potential of
pharmacological ascorbate in cancer treatment will only be realized
with greater understanding of its basic mechanism of action in conjunc-
tion with the appropriate design of clinical trials.
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